Physico-chemical and functional properties of leaf protein concentrates isolated using different techniques : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Riddet Institute, Palmerston North, New Zealand

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Leaf proteins are a potential sustainable protein source for human food as they contain RuBisCo, the most abundant protein in the world (Ellis, 1979). However, these proteins, particularly grasses, are still underutilized due to their undesirable characteristics, such as dark green colour (Di Stefano et al., 2018) and grassy flavour, limiting food applications (Hatanaka, 1996). This thesis studies the effect of different isolation methods on leaf protein concentrates (LPCs) and determines the physico-chemical characteristics of the selected LPCs. In the first part of the study, leaf proteins were extracted through different extraction methods using perennial ryegrass. The result shows that alkali-acid precipitation and alkali-acid precipitation combined with activated carbon (AC) treatment could not remove the green colour and grassy flavour of LPCs. The highest protein content (53.94 % dry basis) and yield (9.64 % of dried grass) of LPC were obtained through alkali-acid precipitation without CaCl₂ treatment. The green colour and grassy flavour were improved using the heat coagulation method without CaCl₂ and NaOH. The protein content was 48.16 % (dry basis); however, a low extraction yield was obtained (1.12 % of dried grass). Although ultrafiltration and chromatography techniques could isolate the brown and colourless LPC, a low yield and protein content were observed. Combining CaCl₂ or AC with the extraction process enhanced the removal of green colour and grassy flavour, but the protein content and extraction yield decreased. Alkali assisted the protein extractability when used with acid precipitation. In contrast, the adverse effects of alkali combined with heat coagulation were obtained. Two leaf protein concentrates (LPCs) were selected to be analysed in the second part: LPC extracted through alkali-acid precipitation without CaCl₂ (AAP-0), and LPC obtained through heat coagulation without CaCl₂ and NaOH (HC-0). AAP-0 had protein solubility higher than HC-0 at pH below 2 and above 5. The protein solubility of AAP-0 was significantly improved (p<0.05) after the treatment of 80 ℃ for 10 minutes combined with pH shift, while the protein solubility of HC-0 was rarely affected using the same treatment. The denaturation temperature and enthalpy of HC-0 were lower than AAP-0. SDS-PAGE results show that the large and small subunits of RuBisCo protein were detected in HC-0, while the large subunit was not detected in AAP-0 due to less protein purity recovered through alkali-acid precipitation. During digestion, AAP-0 had higher solubility than HC-0, whereas its free amino N was lower than HC-0. SDS-PAGE show that AAP-0 had lower digestibility than HC-0 as 10 kDa proteins still appeared in AAP-0 at 180 minutes, while all protein bands were not observed in HC-0.
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